Shigella are bacterial pathogens of man which are the causative agents of bacillary dysentery. Over 200 million cases are reported annually and about 650,000 persons die of shigellosis each year. No effective vaccine exists. Recent bioterrorism concerns increase the importance of understanding Shigella pathogenesis since Shigella has been classified as a category B agent on the NIAID's list of priority pathogens for biodefense research. Shigella is a classic example of an invasive, facultative intracellular pathogen that regulates expression of its virulence genes in response to environmental signals. Our long term objectives have focused on two areas of Shigella pathogenesis: regulation of virulence gene expression and molecular characterization of virulence gene function. The specific aims of this proposal are to : 1) examine the mechanisms by which Shigella regulates virulence gene expression after invasion into the host cell cytoplasm; 2) define the genetic and molecular basis of post invasion phenotypes associated with virulence; and 3) elucidate the mechanism by which components of the type III secretion system interact and recognize virulence proteins which are targeted for secretion. Several models and experimental strategies for testing them are proposed. Genetic and biochemical approaches for measuring DNA-protein interactions will be utilized in the first aim. For aim two, cellular microbiology strategies will be combined with bacterial genetics and molecular biology. For aim three, interactions between components of the type III secretion machinery will be detected by suppressor analysis and use of biochemical techniques. In addition, mutant selection will be applied to define secretion signals recognized by this pathway. This research will fill in important gaps in our knowledge of Shigella pathogenesis. An understanding of the mechanisms that control expression of virulence genes after Shigella invasion can lead to the development of specific drugs that block expression of these genes. Information on how Shigella recognize and secrete virulence factors can also reveal novel targets for the design of new therapeutic agents.